Phosphoinositide 3-kinase in immunological systems

Phosphoinositide 3-kinases (PI3Ks) are an evolutionarily conserved family of signal transducing enzymes. A great variety of stimuli activate PI3K, leading to the transient accumulation of its lipid products in cell membranes. These lipids serve as second messengers to regulate the location and activity of an array of downstream effector molecules. In cells of the mammalian immune system, PI3K is activated by receptors for antigen, cytokines, costimulatory molecules, immunoglobulins and chemoattractants. Signaling via PI3K regulates immune cell proliferation, survival, differentiation, chemotaxis, phagocytosis, degranulation, and respiratory burst. Here we review our current understanding of PI3K signaling in leukocytes.     

Helicobacter pylori regulates TLR4 and TLR9 during gastric carcinogenesis

Objective: To investigated the influence of H. pylori on TLR4 and TLR9 in gastric mucosa during gastric carcinogenesis. Methods: Gastric biopsy specimens were taken from 148 patients and divided into five groups, including normal group (n = 10), chronic superficial gastritis group (n = 35), atrophy/intestinal metaplasia group (n = 35), dysplasia group (n = 34) and gastric carcinoma group (n = 34). Immunohistochemistry was used to detect the expression of TLR4 and TLR9. Geimsa staining and rapid urea test were used for determine H. pylori infection. Results: TLR4 was detected in gastric epithelium and monocytes/macrophages in superficial gastritis, atrophy/intestinal metaplasia, dysplasia or carcinoma. TLR9 was mainly accentuated in monocytes/macrophages. TLR4 positive cells in epithelium and in monocytes/macrophages with H. pylori infection were much more than those without H. pylori infection. Similar results were also found in TLR9. When gastric epithelium was accompanied with H. pylori infection, TLR4 was significant higher in superficial gastritis and atrophy/intestinal metaplasia groups compared with dysplasia and carcinoma groups. When gastric epithelium was infected by H. pylori, TLR9 was significant higher in carcinoma group compared with superficial gastritis, atrophy/intestinal metaplasia and dysplasia. TLR4 and TLR9 show significant correlation with the severity of inflammation. Conclusions: H. pylori infection was associated with increased expression of TLR4 and TLR9 in gastric mucosa. In superficial gastritis and atrophy/intestinal metaplasia the inflammation was predominately mediated by TLR4, while in gastric cancer the inflammation was mainly mediated by TLR9.     

Phosphoinositide 3-kinase: from viral oncoprotein to drug target

The catalytic subunit p110alpha of the phosphoinositide 3-kinase (PI3K) and the serine-threonine protein kinase Akt have been extensively studied as retroviral oncoproteins. The experimental tools developed with the retroviral vectors are now being applied to PI3K mutations in human cancer. The most frequently occurring mutants of p110alpha are oncogenic in vitro and in vivo, show gain of enzymatic function, activate Akt, and their oncogenic activity is sensitive to rapamycin. The related isoforms p110beta, gamma and delta induce oncogenic transformation as wild-type proteins. Mutated p110alpha proteins are ideal drug targets. Identification of small molecule inhibitors that specifically target these mutant proteins is a realistic and urgent goal.     

Monocyte p110alpha phosphatidylinositol 3-kinase regulates phagocytosis, the phagocyte oxidase, and cytokine production

Mononuclear phagocytes are critical modulators and effectors of innate and adaptive immune responses, and PI-3Ks have been shown to be multifunctional monocyte regulators. The PI-3K family includes eight catalytic isoforms, and only limited information is available about how these contribute to fine specificity in monocyte cell regulation. We examined the regulation of phagocytosis, the phagocyte oxidative burst, and LPS-induced cytokine production by human monocytic cells deficient in p110alpha PI-3K. We observed that p110alpha PI-3K was required for phagocytosis of IgG-opsonized and nonopsonized zymosan in differentiated THP-1 cells, and the latter was inhibitable by mannose. In contrast, p110alpha PI-3K was not required for ingestion serum-opsonized zymosan. Taken together, these results suggest that FcgammaR- and mannose receptor-mediated phagocytosis are p110alpha-dependent, whereas CR3-mediated phagocytosis involves a distinct isoform. It is notable that the phagocyte oxidative burst induced in response to PMA or opsonized zymosan was also found to be dependent on p110alpha in THP-1 cells. Furthermore, p110alpha was observed to exert selective and bidirectional effects on the secretion of pivotal cytokines. Incubation of p110alpha-deficient THP-1 cells with LPS showed that p110alpha was required for IL-12p40 and IL-6 production, whereas it negatively regulated the production of TNF-alpha and IL-10. Cells deficient in p110alpha also exhibited enhanced p38 MAPK, JNK, and NF-kappaB phosphorylation. Thus, p110alpha PI-3K appears to uniquely regulate important monocyte functions, where other PI-3K isoforms are uninvolved or unable to fully compensate.     

Phosphoinositide 3-kinase and its targets in B-cell and T-cell signaling

Phosphoinositide 3-kinase (PI3K) activation is essential for lymphocyte proliferation driven by receptors for antigen, costimulatory ligands and cytokines. The lipid products of PI3K contribute to the assembly of membrane-associated signaling complexes by promoting recruitment of selected proteins from the cytoplasm. Many proteins possess domains that are able to bind selectively to PI3K products. Different 'PI3K effector' proteins are coupled to distinct biological responses, depending on cell type and on the receptor that is engaged. In B cells and T cells, Tec-family tyrosine kinases and Akt serine/threonine kinases are emerging as crucial mediators of proliferation and survival signals downstream of PI3K. Of particular interest is recent evidence that PI3K signaling controls increases in lymphocyte size and metabolic activity that accompany cell cycle progression.     

Phosphoinositide 3-kinase signalling in lung disease: leucocytes and beyond

The family of lipid kinases termed phosphoinositide-3-kinase (PI3K) is known to contribute at multiple levels to innate and adaptive immune responses, and is hence an attractive target for drug discovery in inflammatory and autoimmune disease, including respiratory diseases. The development of isoform-selective pharmacological inhibitors, targeted gene manipulation and short interfering RNA (siRNA) target validation have facilitated a better understanding of the role that each member of this family of kinases plays in the physiology and pathology of the respiratory system. In this review, we will evaluate the evidence for the roles of specific PI3K isoforms in the lung and airways, and discuss their potential as targets for novel drug therapies.     

Phosphoinositide 3-kinase gamma is an essential amplifier of mast cell function

Mast cells are key regulators in allergy and inflammation, and release histamine upon clustering of their IgE receptors. Here we demonstrate that murine mast cell responses are exacerbated in vitro and in vivo by autocrine signals through G protein-coupled receptors (GPCRs) and require functional phosphoinositide 3-kinase gamma (PI3Kgamma). Adenosine, acting through the A(3) adenosine receptor (A(3)AR) as well as other agonists of G(alphai)-coupled GPCRs, transiently increased PtdIns(3,4,5)P(3) exclusively via PI3Kgamma. PI3Kgamma-derived PtdIns(3,4,5)P(3) was instrumental for initiating a sustained influx of external Ca(2+) and degranulation. Mice lacking PI3Kgamma did not form edema after intradermal injection of adenosine and when challenged by passive systemic anaphylaxis. PI3Kgamma thus relays inflammatory signals through various G(i)-coupled receptors and is central to mast cell function.     

Role of actin polymerization in monocyte phagocytosis of yeast cells effect of cytochalasin B

The role of the cortical actomyosin-like contractile system in monocyte phagocytosis was studied by means of inhibition with cytochalasin B. Monocyte phagocytosis of yeast cells was assayed with a fluorescence extinction technique of surface-located phagocytosis which distinguishes between adherence and ingestion phases of total phagocytosis. Cytochalasin B, 10 micrograms/ml, inhibited monocyte phagocytosis by 20-30%. The cytochalasin inhibition was significant and restricted to the ingestion phase. The finding supported the hypothesis that the contractile structures of the cell have a contributory role in IgG-mediated monocyte phagocytosis on a surface. It is suggested that the cytochalasin effect reflected the role of chemotaxis in monocyte phagocytosis assayed by the present technique.     

Tyrosine phosphatase MptpA of Mycobacterium tuberculosis inhibits phagocytosis and increases actin polymerization in macrophages

Protein tyrosine phosphatases from several microorganisms have been shown to play a role as virulence factors by modifying the phosphorylation/dephosphorylation equilibrium in cells of their host. Two tyrosine phosphatases, MptpA and MptpB, secreted by Mycobacterium tuberculosis, have been identified. Expression of MptpA is upregulated upon infection of monocytes, but its role in host cells has not been elucidated. A eukaryotic expression vector containing the mptpA cDNA has been transfected into macrophages. We report that MptpA reduced phagocytosis of mycobacteria, opsonized zymosan or zymosan, but had no effect on phagocytosis of IgG-coated particles. We also noted that the presence of F-actin at the surface of phagosomes containing opsonized zymosan was significantly increased in cells expressing MptpA. In the presence of recombinant MptpA, the process of actin polymerization at the surface of isolated phagosomes was increased; this was not the case in the presence of the phosphatase-dead mutant MptpA(C11S). MptpA had no effect when IgG-coated particles were present inside isolated phagosomes. These results indicate that, like other tyrosine phosphatases of pathogens, MptpA plays a role in phagocytosis and actin polymerization. However, MptpA had no effect on IgG particles, suggesting that its putative substrate(s) is not linked to the signaling pathways of Fcgamma receptors.     

Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation

Shiga toxin-producing enterohemorrhagic E. coli infections cause bloody diarrhea, which may progress to life-threatening complications such as the hemolytic-uremic syndrome (HUS). HUS patients frequently have elevated levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) detectable in urine. Thus, sequelae may develop following the localized production of proinflammatory cytokines within the kidneys. A possible source of these cytokines are macrophages, which respond to the toxins by producing TNF-alpha. We have shown previously that THP-1 cells produce soluble TNF-alpha in response to the toxins, whose production requires host-cell tyrosine-kinase activity and toxin-enzymatic activity. To further examine signaling pathways involved in TNF-alpha expression, we determined that JNK1 and -2 and p38, but not ERK1 or -2, were phosphorylated following toxin exposure. Blockade of p38 activation reduced TNF-alpha production following Shiga toxin 1 treatment. Finally, we present a model of the ribotoxic stress response triggered in human macrophages by Shiga toxins.     

Stimulation by target cell membrane lipid of actin polymerization and phagocytosis by Entamoeba histolytica.

The identity of molecules of mammalian target cells that stimulate contact-dependent attack by Entamoeba histolytica was sought using human erythrocytes (RBC) as a model. Protein-free liposomes prepared from RBC membrane lipids stimulated the same rapid E. histolytica actin polymerization and phagocytosis as did whole target cells. Liposomes constructed from the major phospholipids of RBC stimulated these responses but only if a negatively charged phospholipid was included. The addition to these liposomes of digalactosyl diglyceride significantly enhanced their stimulatory activity. The results demonstrate that ligands that trigger attack-related responses by E. histolytica reside in the target cell membrane lipid fraction and suggest roles for both glycolipid and phospholipid components.     

Light microscopic and ultrastructural evidence of in vivo phagocytosis of Helicobacter pylori by neutrophils

Helicobacter pylori is believed to cause chronic active gastritis. Infection/colonization of the gastric mucosal surface induces a mucosal inflammatory reaction in the form of lymphocytic aggregates, plasma cells and, particularly, neutrophils, which may, in turn, damage the mucosal epithelium. In vitro studies demonstrate that, in culture, the bacilli are readily phagocytosed by neutrophils, this evoking a neutrophilic oxidative burst. However, it has been claimed that neutrophils do not phagocytose H. pylori in vivo. In this study of 19 endoscopic biopsies of gastric mucosa with H. pylori-associated gastritis, Cresyl violet staining for light microscopy and electron microscopy are used to demonstrate that, in vivo, neutrophils actively phagocytose and destroy the bacilli in the epithelial intercellular space and in the mucin on the surface of the mucosa. Direct contact of neutrophils with H. pylori was observed in 17 of 17 cases by light microscopy and in 4 of 15 cases by electron microscopy. Phagocytosis by neutrophils was seen in 14 of 17 cases by light microscopy and in 3 of 1 5 cases by electron microscopy. It was most evident in the surface mucus coat where "wolf packs" of neutrophils were seen attacking the microbes. Ultrastructurally, neutrophil phagolysosomes contained both intact and partially digested bacteria, convincing evidence that the primary function of neutrophils in chronic active gastritis is to destroy H. pylori organisms. This study leaves open the question of whether, or how, neutrophils damage the gastric mucosa.     

The novel acute phase protein, IHRP, inhibits actin polymerization and phagocytosis of polymorphonuclear cells

OBJECTIVE AND DESIGN: In the present study, the involvement of the binding of IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) and actin in phagocyte activity was investigated. MATERIALS AND METHODS: The actin polymerization and the phagocytic activity of the polymorphonuclear (PMN) cells were studied in the presence of IHRP. RESULTS: IHRP inhibited the polymerization of actin and the phagocytic activity of the PMN cells. CONCLUSION: 1) IHRP may bind to actin released from the damaged cells and suppress its toxic action by preventing the formation of actin fibril. 2) IHRP may bind to cell surface actin on PMN cells and inhibit their phagocytic activities. 3) From these results, IHRP may act as an anti-inflammatory protein.     

L-arginine availability regulates inducible nitric oxide synthase-dependent host defense against Helicobacter pylori

Helicobacter pylori infection of the stomach causes an active immune response that includes stimulation of inducible nitric oxide (NO) synthase (iNOS) expression. Although NO can kill H. pylori, the bacterium persists indefinitely, suggesting that NO production is inadequate. We determined if the NO derived from iNOS in macrophages was dependent on the availability of its substrate, L-arginine (L-Arg). Production of NO by H. pylori-stimulated RAW 264.7 cells was dependent on the L-Arg concentration in the culture medium, and the 50% effective dose for L-Arg was 220 microM, which is above reported plasma L-Arg levels. While iNOS mRNA induction was L-Arg independent, iNOS protein increased in an L-Arg-dependent manner that did not involve changes in iNOS protein degradation. L-lysine, an inhibitor of L-Arg uptake, attenuated H. pylori-stimulated iNOS protein expression, translation, NO levels, and killing of H. pylori. While L-Arg starvation suppressed global protein translation, at concentrations of L-Arg at which iNOS protein was only minimally expressed in response to H. pylori, global translation was fully restored and eukaryotic translation initiation factor alpha was dephosphorylated. H. pylori lacking the gene rocF, which codes for a bacterial arginase, induced higher levels of NO production by increasing iNOS protein levels. When murine gastric macrophages were activated with H. pylori, supraphysiologic levels of L-Arg were required to permit iNOS protein expression and NO production. These findings indicate that L-Arg is rate limiting for iNOS translation and suggest that the levels of L-Arg that occur in vivo do not permit sufficient NO generation by the host to kill H. pylori.     

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.     

Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells

Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.     

Chemotaxis plays multiple roles during Helicobacter pylori animal infection

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as specific gastric cancers. H. pylori infects approximately 50% of the world's population, and infections can persist throughout the lifetime of the host. Motility and chemotaxis have been shown to be important in the infection process of H. pylori. We sought to address the specific roles of chemotaxis in infection of a mouse model system. We found that mutants lacking cheW, cheA, or cheY are all nonchemotactic and infect FVB/N mice with an attenuated phenotype after 2 weeks of infection. If infections proceeded for 6 months, however, this attenuation disappeared. Histological and culture analysis revealed that nonchemotactic mutants were found only in the corpus of the stomach, while the wild type occupied both the corpus and the antrum. Further analysis showed that nonchemotactic H. pylori isolates had an increased 50% infectious dose and were greatly outcompeted when coinfected with the wild type. If nonchemotactic mutants were allowed to establish an infection, subsequent infection with the wild type partially displaced the nonchemotactic mutants, indicating a role for chemotaxis in maintenance of infection. The data presented here support four roles for chemotaxis in H. pylori mouse infections: (i) establishing infection, (ii) achieving high-level infection, (iii) maintaining an infection when there are competing H. pylori present, and (iv) colonizing all regions of the stomach.     

Helicobacter pylori regulates iNOS promoter by histone modifications in human gastric epithelial cells

Inducible nitric oxide synthase (iNOS) expression is altered in gastrointestinal diseases. Helicobacter pylori (Hp) infection may have a critical role in iNOS disregulation. We undertook this study to investigate possible chromatin changes occurring early during iNOS gene activation as a direct consequence of Hp-gastric cells interaction. We show that Hp infection is followed by different expression and chromatin modifications in gastric cells including (1) activation of iNOS gene expression, (2) chromatin changes at iNOS promoter including decreased H3K9 methylation and increased H3 acetylation and H3K4 methylation levels, (3) selective release of methyl-CpG-binding protein 2 from the iNOS promoter. Moreover, we show that Hp-induced activation of iNOS is delayed, but not eliminated, by the treatment with LSD1 inhibitors. Our data suggest a role for specific chromatin-based mechanisms in the control of human iNOS gene expression upon Hp exposure.